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Abstract

Background

Paxillin (PXN) has been found to be aberrantly regulated in various malignancies and
involved in tumor growth and invasion. The clinicopathological and prognostic significance
of PXN in gastric cancer is still unclear.

Methods

The expression of PXN was determined in paired gastric cancer tissues and adjacent
normal tissues by Western blotting and real-time PCR. Immunohistochemistry was performed
to detect the expression of PXN in 239 gastric cancer patients. Statistical analysis
was applied to investigate the correlation between PXN expression and clinicopathological
characteristics and prognosis in patients. Additionally, the effects of PXN on gastric
cancer cell proliferation and migration were also evaluated.

Conclusions

PXN plays an important role in tumor progression and may be used as a potential prognostic
indicator in gastric cancer.

Keywords:

Gastric cancer; Paxillin; Tumor progression; Prognosis

Background

Gastric cancer is among the most frequently diagnosed cancer and the second leading
cause of cancer-related mortality worldwide [1,2]. In spite of recent improvement in the clinical treatment of gastric cancer, patients
with advanced stage of gastric cancer still have poor survival. Surgical resection
is still the only curative therapy available for this disease [3]. Under this circumstance, there is an urgent need to better understand the biological
mechanism of this neoplasm so as to guide patient management and develop novel therapeutic
strategies.

The paxillin (PXN) gene was first identified as a tyrosine-containing protein in cells
transformed by the src oncogene [4] and encodes for a focal adhesion molecule of 68 kD [5]. PXN functions as an adaptor protein that coordinates multiple signals from integrins,
growth factors and cell surface receptors [6]. By these protein-protein interactions, PXN regulates diverse physiological process,
such as gene expression, matrix organization, tissue remolding, cell proliferation
and survival, cell motility and metastasis [6-8]. In addition to the interactions with cytoskeleton proteins, PXN could also bind
to several oncogenic proteins, such as v-Src, E6 and BCR-ABL [9-13]. Such proteins could use PXN as a docking site or as a substrate to interrupt or
mislead the normal adhesion and growth factor signaling pathways that are essential
for controlled cellular growth and migration [5]. PXN has been found to be involved in many tumor types. A previous study reported
that PXN could inhibit lung cancer proliferation and motility [14]. However, more recent studies found PXN was overexpressed and acted as an pro-oncogene
in a variety of tumors, including non-small cell lung cancer, colorectal cancer, prostate
cancer and cervical carcinoma [15-20]. In gastric cancer, Li et al. reported that PXN (tyr118) phosphorylation was a key factor for fibronectin-stimulated
invasiveness of AGS cells [21]. However, the clinicopathological and prognostic role of PXN in gastric cancer is
still unclear.

In this study, we detected the PXN mRNA and protein level in 30 paired tumor tissues
and adjacent normal tissues and found that PXN was frequently up-regulated in tumor
tissues. In addition, the expression of PXN was associated with poor prognosis in
a large cohort of 239 patients. Furthermore, ectopic expression/knockdown of PXN could
promote/inhibit cell proliferation and migration in gastric cancer cells.

Materials and methods

Human tissue specimens and cell lines

This study was approved by the ethics committee of Sun Yat -sen University Cancer
Center and written informed consents for using the samples for research purpose were
obtained from all the patients before surgery. We collected 239 paraffin-embedded,
archived tissue samples from patients who underwent surgery in Sun Yat-sen University
Cancer Center (Guangzhou, China) from 2004 to 2008. Moreover, we obtained paired fresh
gastric cancer tissues and adjacent nontumorous tissues from 30 of the 239 patients
and kept in liquid nitrogen until use. All the patients had a histologically confirmed
diagnosis of gastric cancer after resection. Tumor stage was determined according
to the 7th edition of the International Union Against Cancer (UICC) on Tumor-Node-Metastasis
(TNM) staging system. All the patients were followed-up regularly every three months
after surgery with a median follow-up time of 32.5 months (range from 4 to 75 months).
All the patients did not receive any pre-operative treatment. The patients who received
adjuvant chemotherapy after surgery were based on 5-FU, platinum or taxol regimens.
Relevant clinicopathological information including age, gender, tumor size, tumor
depth, lymph node invasion, distant metastasis, differentiation status, TNM stage
and treatment strategies were obtained from patients’ medical files.

RNA extraction and real-time quantitative RT-PCR analysis

Total RNA was extracted from the tissues or cells using Trizol reagent (Invitrogen,
Carlsbad, CA, USA) and complementary DNA (cDNA) was synthesized with 2 μg of total
RNA by using M-MLV transcriptase (Promega, Madison, WI). Real-time PCR was performed
with an ABI PRISM® 7500 Fast Real-time PCR System (Applied Biosystems, CA, USA) and
a SYBR Premix Ex Taq™ kit (Takara, Japan); β-actin expression was used as a reference.
The following temperature profiles were used: initial heating at 95°C for 10 min,
followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s,
and extension at 65°C for 10 s. The primers used were:

PXN forward: 5′-ACGTCTACAGCTTCCCCAACAA-3′;

PXN reverse: 5′-AGCAGGCGGTCGAGTTCA-3′;

β-actin forward: 5′-TGGATCAGCAAGCAGGAGTA-3′;

β-actin reverse: 5′-TCGGCCACATTGTGAACTTT-3′.Data were analyzed using the 2-△△ct method.

Western blot analysis

Total cellular proteins were extracted from tissues or cells and separated by SDS-PAGE
gels, Western blot analysis was performed according to a standard method as previously
described [22]. For immunoblotting of PXN, a rabbit PXN antibody (1:1000, Cell Signaling Technology,
USA) was used, and an anti-GAPDH antibody (1:2000; Santa Cruz Biotechnology, USA)
was used as loading control.

Immunohistochemistry (IHC) analysis

IHC analysis of PXN was performed according to a previously described method [23]. Briefly, the formalin-fixed, paraffin-embedded tissue samples were cut into 4 μm
slides, dewaxed in xylene, rehydrated with graded of alcohols, and then treated with
3% hydrogen peroxide to block endogenous peroxidase activity. The slides were boiled
in 0.01 mol/L sodium citrate buffer (pH 6.0) in a microwave oven to retrieve tissue
antigens. Tissue samples were pretreated with 10% normal goat serum to inhibit non-specific
staining and incubated at 4°C with a primary antibody (ab#32084, abcam) over night.
Tissue sections were then washed with PBST, treated with an anti-rabbit secondary
antibody, incubated with streptavidin horseradish peroxidase complex, and finally
developed using diaminobenzidine tetrahydrochloride (DAB).

To assess the expression of PXN, we qualified and scored both the extent and intensity
of immunoreactivity. In this study, the scores of the extent of staining were evaluated
according to the percentage of cells that had positive immunoreactivity in every microscopic
field of view: 0, <25%; 1, 25%-50%; 2, 50%-75%; 3, 75%-100%. The scores of IHC intensity
ranging from 0 to 3 were determined as follows: 0, negative staining; 1, weak staining;
2, moderate staining; 3, strong staining. By multiplying the scores for extent and
intensity, a total score (range, 0 to 9) was achieved. PXN expression level was considered
high with scores of ≥ 4 and low with scores of < 4.

Cell transfection

For overexpression of endogenous PXN, the coding sequence of PXN was amplified and
subcloned into the pcDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, USA) according to
the manufacturer’ instructions. AGS cells were then transfected with a negative control
vector or a PXN expressing plasmid using lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA). To knockdown endogenous PXN expression in cells, small interfering RNA (siRNA)
duplex oligonucleotides targeting human PXN mRNA (si-PXN) was obtained from Ribobio
(Guangzhou, China). The targeting sequences were: si-PXN#1: GCAGCAACCTTTCTGAACT; si-PXN#2:
GTGTGGAGCCTTCTTTGGT. In the present study, we used si-PXN#1 as it could effectively
reduced endogenous PXN expression in our preliminary experiments. The target sequence
for scrambled siRNA was 5′-GTCTCCACGCGCAGTACATTT-3′. SGC7901 cells were transfected
with si-PXN or scramble siRNA according to the manufacturer’s instructions.

Cell proliferation assays

The 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide (MTT) assay was
performed to test cell proliferation following a method as previously described [24]. Cells were seeded in a 96-well plate at 1 × 103 cells/well, the spectrophotometric absorbance was measured for each sample at 490 nm,
all the experiments were performed in triplicate and repeated for 3 times, and the
average was calculated.

For the colony formation assay, cells (500/well) were seeded in a six-well plate and
cultured for 14 days at 37°C with 5% CO2 humidified air. Colonies were stained with 0.1% crystal violet (1 mg/ml) and the
numbers of colonies containing more than 50 cells were counted. The experiment was
performed in triplicate and repeated for three times.

In vitro cell migration assay

The cell migratory capacity was determined using transwell chambers (BD Biosciences)
according to a method previously described [25]. Briefly, cells (1 × 105/well) were suspended in 100 μl serum-free medium and then added to the upper chamber
of the inserts, RPMI 1640 medium (GIBCO) containing 10% FBS (500 μl) was added to
the lower chamber as the chemotactic factor. After culture for 22 hours, non-migrated
cells on the upper surface were removed gently with a cotton swab and cells that migrated
to the lower side of the department were fixed and dyed with 0.1% crystal violet.
The numbers of migrated cells were calculated by counting five different views under
the microscopy. The experiment was performed in triplicate and repeated for three
times.

Statistical analysis

All the data were presented as mean ± SD, a Student t-test or Chi-square test was
employed to compare the differences as appropriate. Survival analysis was performed
using the Kaplan-Meier method and the log-rank test. Multivariate analysis with Cox
proportional hazards model was used to investigate independent prognostic factors.
All P-values were two-sided, and a P-value of < 0.05 was considered statistically significant. Statistical analysis was
performed by the SPSS software package (version 16.0, SPSS Inc) or GraphPad prism
5.

Results

PXN is up-regulated in gastric cancer tissues and cell lines

The protein and mRNA level of PXN was evaluated in gastric cancer tissues and cell
lines. In paired primary gastric cancer tissues and adjacent nontumorous tissues,
Western blot analysis revealed overexpression of PXN in cancer tissues compared with
adjacent nontumorous tissues (Figure 1A). The level of PXN mRNA was increased in 27 of 30 (P < 0.001) cancer tissues in comparison with that of nontumorous tissues (Figure 1B). Moreover, Western blot analysis showed the expression levels of PXN were higher
in gastric cancer cell lines than that of normal gastric epithelial cell line GES-1
(Figure 1C). Consistent with the level of protein, real-time PCR analysis confirmed PXN mRNA
level was elevated in all five gastric cancer cell lines (Figure 1D).

Overexpression of PXN is associated with adverse tumor phenotype and poor prognosis
in gastric cancer

We further investigated the expression of PXN in 239 paraffin-embedded gastric cancer
tissues by IHC. As shown in Figure 2, PXN protein was mainly located in the cytoplasm of tumor cells. Positive staining
was detected in 186 of 239 (77.8%) patients. One hundred and fifty-two patients were
possessed with low PXN expression while the other eighty-seven with high PXN expression
based on the IHC scores. The associations between PXN expression and clinical and
pathologic parameters were summarized in Table 1. High PXN expression was significantly correlated with distant metastasis (P = 0.001), TNM stage (P = 0.021) and survival status (P = 0.004). However, there was no association between PXN expression level and age,
gender, tumor depth, differentiation status and treatment. Kaplan-Meier analysis with
log-rank test was employed to investigate the prognostic role of PXN for gastric cancer
patients. Patients with high PXN expression had significantly worse overall survival
than patient with low PXN expression (Figure 3). In addition, Univariate analysis revealed high PXN expression is a risk factor
of death in gastric cancer patients (HR = 2.05, 95%CI, 1.47-2.86; P < 0.001, Table 2). Besides, other factors including lymph node invasion, distant metastasis and TNM
stage were also correlated with overall survival as demonstrated by univariate analysis
(Table 2). However, age, gender, differentiation status, treatment and tumor depth were not
prognostic indicators in this study. Multivariate analysis indicated only distant
metastasis and PXN expression were independent prognostic factors for gastric cancer
patients (P < 0.001 and P = 0.020, respectively, Table 2).

PXN promotes tumor growth and proliferation in vitro

We further investigated the role of PXN on the tumor cell growth and proliferation
through gain-of-function and loss-of-function analysis. AGS cells were transfected
with pcDNA3.1(+)-PXN to overexpress PXN (Figure 4A). The effects of PXN on cell growth and proliferation were then evaluated by MTT
and colony formation assays. The results showed that ectopic expression of PXN significantly
promoted the viability of AGS cells (Figure 4B, P = 0.017). The colony formation assay also indicated the number of colonies was markedly
increased in PXN overexpressing cells compared with control cells (Figure 4D and E, P = 0.026). The impact of PXN on cellular proliferation was further confirmed by performing
MTT and colony formation assays after PXN knockdown in SGC7901 cells (Figure 4A). As indicated in Figure 4C, F and G, compared with that of control cells, knockdown of PXN significantly inhibited
the growth rate and colony formation ability of SGC7901 cells (P = 0.019 and P = 0.011, respectively).

PXN promotes cell migration in vitro

As PXN expression is associated with distant metastasis in gastric cancer patients,
we then evaluated the potential role of PXN on cellular migration by transwell assays.
AGS cells were transfected with PXN overexpressing or control plasmid and seeded in
the chamber, and their migratory abilities were determined 24 hours later. The results
showed ectopic expression of PXN significantly increased the migratory capacity of
AGS cells (Figure 5A and B, P = 0.013). On the contrary, knockdown of PXN dramatically reduced the migrated cell
number of SGC7901 (Figure 5C and D, P = 0.032).

Discussion

We have previously found that PXN is up-regulated in colorectal cancer and associated
with aggressive tumor phenotypes [26]. However, the potential clinical and pathologic role of PXN in gastric cancer is
still unknown. In this study, firstly, we detected the expression of PXN in gastric
cancer tissues and cell lines. The results showed that PXN was frequently up-regulated
in gastric cancer tissues and cell lines compared with adjacent nontumorous tissues
or normal gastric mucosa cells. In addition, in a large cohort of gastric cancer patients,
overexpression of PXN was significantly associated with aggressive tumor characteristics,
such as distant metastasis and advanced TNM stage. Moreover, high expression of PXN
was correlated with poor overall survival. Multivariate analysis demonstrated that
PXN expression was an independent prognostic factor for gastric cancer patients. These
results demonstrated that overexpression of PXN was commonly observed in gastric cancer.
PXN might serve as an useful prognostic indicator for patients.

Several studies have reported the clinical and pathological significance of PXN in
other tumor types. For example, up-regulation of PXN is found in non-small cell lung
cancer [15] and prostate cancer [27]. Expression of PXN is associated with adverse pathologic characteristics in hepatocellular
carcinoma [28]; PXN protein level is correlated with advanced clinical stage and distant metastasis
in salivary adenoid cystic carcinoma [29]; amplification of PXN is frequently observed in high risk lung cancer [17]. Our findings in gastric cancer are in lines with these results. However, further
studies are needed to confirm the clinical role of PXN in gastric cancer.

PXN is a focal adhesion molecule that involved in signaling transduction and cellular
migration, it can increase the adhesion between tumor cells and the surrounding extracellular
matrix and molecules, thereby promotes or inhibits the tumor cell motilities. The
effect of PXN on cell spreading is mainly regulated by tyrosine/serine phosphorylation
[30,31]. For instance, in breast cancer, cell migration and invasion stimulated by breast
tumor kinase (Brk) is mediated through PXN phosphorylation [32], PXN is the substrate of Brk and functions as a “platform” of Brk. In colon cancer,
phosphorylation of PXN at tyrosines 31 and 118 site is essential for pressure-induced
cellular metastasis and adhesion [33]. In this study, we found that ectopic expression of PXN stimulated tumor proliferation
and migration whereas knockdown of PXN suppressed cellular growth and motility in
gastric cancer cells. Our results are similar to what observed in colorectal cancer,
in which Jun et al. found that ectopic expression of PXN could increase cell migration, invasion and
adhesion abilities whereas knockdown of PXN expression by small interfering RNA suppressed
these capacities [19]. Taken together, these results suggest that PXN plays an important role in the growth
and metastasis of gastric cancer.

Conclusions

In conclusion, our study revealed a cell adhesion protein PXN is frequently up-regulated
in gastric cancer tissues and cell lines. Overexpression of PXN is associated with
aggressive tumor phenotypes and adverse overall survival, thus implicating PXN might
serve as an useful prognostic indicator. Ectopic expression/knockdown of PXN promotes/inhibits
tumor growth and migration, which indicates that PXN may play an important role in
the progression and metastasis of gastric cancer. However, further study is needed
to investigate the underlying mechanism involved in PXN regulation in gastric cancer.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

CDL conceived of the study, performed the Western blotting analysis, IHC analysis,
molecular studies and drafted the manuscript. WZQ, RC, ZZL and WDS collected the clinical
data and tissue samples. LHY, WF, QMZ, BL, ZDS, WFH and LYH performed the statistical
analysis. XRH participated in the design of the study and helped to draft the manuscript.
All authors read and approved the final manuscript.

Acknowledgements

This study was sponsored by the grant from National High-tech R&D Program (863 Program),
China (No.2012AA02A506) and The Science and Technology Department of Guangdong Province,
China (No. 2012B031800088).